1. Field of the Invention
This invention relates to biotechnology generally and, more specifically, to an ex vivo animal or challenge model as a method to measure protective circuitry directed against parasites and vaccines shown to be protective in the method.
2. State of the Art
Only a few vaccines against parasites are commercially available. Most of these vaccines are based on attenuated live parasites that induce natural, protective immunity and cause less severe pathological damage. These parasite vaccines include one directed against Dictyocaulus viviparus (e.g., Dictol, Glaxo), undoubtedly the most successful anti-parasite vaccine, and analogous therewith a vaccine against Dictyocaulus filaria, the lung worm in sheep (Sharma et al. 1988). These vaccines are based on live but irradiated third-stage larvae (Peacock and Pointer 1980). Another attenuated vaccine is directed against the hookworm Ancylostoma caninum in dogs. However, this vaccine has been marketed only for a short time in the USA; marketing was discontinued because the American veterinary profession did not accept this live vaccine (Urquhart 1980). An attenuated vaccine against Babesia bovis has been in use for nearly a century in Australia (Purnell 1980) and a dead vaccine based on metabolic products named “Pirodog” is used to vaccinate dogs against B. canis (Moreau 1986).
Vaccination trials in sheep with a recombinant vaccine against the tape worm Taenia ovis (Johnson et al. 1989) and the concealed antigen H11 from Haemonchus contortus (Newton 1995, review) have been performed successfully. A trial with the SPf66 malaria vaccine in Africa has recently been completed. The efficiency against clinical malaria in areas of high transmission was 31% and the product appeared to be safe. However, because it is not fully understood how SPf66 mediates protection, the development of improved vaccines is hampered (Tanner et al. 1995, review).
Problems of developing anti-parasite vaccines are abundant. Parasites have complex life cycles and each stage expresses different sets of antigens. Moreover, the different stages are often associated with different sites in the body. For most parasites little is known about the immune mechanisms involved in natural immunity and about the stage of the parasite inducing this immunity.
Most often, no reproducible animal model is available to study these mechanisms, thereby blocking a new approach in vaccine development. As mentioned above, most available vaccines are based on attenuated live parasites. These vaccines can sometimes be successful because the “vaccine parasites” follow the correct route of infection and deliver a wide array of stage-specific antigens. However, such vaccines must challenge the acceptance of the public (e.g., Ancylostoma caninum vaccine), especially when they are for human use (e.g., Schistosoma mansoni vaccine, Taylor et al. 1986). Moreover, live vaccines, in general, have a short shelf-life and are relatively expensive. From this perspective there is an obvious need for vaccines that are based on (recombinant) proteins derived from the parasite. However, the identification of such protective proteins meets a great number of difficulties, as shown below as an example for Fasciola hepatica. 
The trematode parasite Fasciola hepatica mainly infects cattle and sheep. Sometimes also humans get infected. The parasite causes considerable economic losses in, for example, western Europe, Australia and South America. The metacercariae of Fasciola hepatica enter its host by the oral route, penetrate the gut wall within four to seven hours (Dawes 1963; Burden et al. 1981; Burden et al. 1983; Kawano et al. 1992) and migrate through the peritoneal cavity towards the target organ, the liver. Oral infection of cattle results in almost complete protection against a challenge, whereas sheep often die from an infection and do not acquire natural immunity. Both the natural host (cattle) and the animal model (rat) acquire natural immunity after infection (Doy and Hughes 1984; Hayes, Bailer and Mitrovic 1973). Therefore, rats are often used to study resistance in cattle. In the rat a large part of natural immunity is expressed in the gut mucosa, the porte d'entree of the parasite. In immune rats, about 80% of the challenge newly excysted juvenile stages (NEJs) is eliminated in the route from the gut lumen to the peritoneal cavity (Hayes and Mitrovic 1977; Rajasekariah and Howell 1977; Doy, Hughes and Harness 1978/1981; Doy and Hughes 1982; Burden et al. 1981/1983). Based on natural immunity, a vaccine based on irradiated Fasciola gigantica metacercariae was developed for cattle (Bitakaramire 1973). In the seventies and eighties many vaccination experiments have been performed with antigen extracts of adult and juvenile flukes (Haroun and Hillyer 1980, review). However, these studies lead to conflicting or disputable results. For example, subcutaneous or intramuscular injection of rats with adult or juvenile fluke extracts did not result in protection (Oldham and Hughes 1982; Burden et al. 1982; Oldham 1983). Adult fluke extracts given intraperitoneally in Freund complete adjuvant (FCA) or incomplete Freund adjuvant (IFA) resulted in about 50% protection (Oldham and Hughes 1982; Oldham 1983). Using very high antigen doses of Bordetella pertussis as additional adjuvant this protection reached 80% to 86% (Oldham and Hughes 1982; Oldham 1983). Extracts of four-week-old juveniles given intraperitoneally in AlOH3 did not induce protection in the studies of Pfister et al. (1984/85), whereas 16-day old juvenile extracts provided 86% protection in mice, without the use of adjuvant (Lang and Hall 1977). Subcutaneous sensitization of cattle with sonicated 16-day-old juveniles resulted in more than 90% protection (Hall and Lang 1978). Intramuscular injection of calves with an isolated fraction from adult Fasciola hepatica (FhSmIII), with an immunogenic 12 kD protein as major component, resulted in 55% protection (Hillyer et al. 1987).
Since 1990, several Fasciola hepatica vaccine candidate antigens have been isolated and/or produced. Most of these antigens are derived from adult flukes and share homology with Schistosoma mansoni antigens. Glutathion S-transferases (GST) are enzymes amongst others active in the cellular detoxification system. Immunization of sheep (n=9) with GST purified from adult Fasciola hepatica, injected s.c. in FCA, with a boost immunization 4 weeks later in IFA, resulted in 57% protection (Sexton et al. 1990). Immunization of rats with GST provided no protection (Howell et al. 1988). Vaccination trials in cattle performed by Ciba Animal Health Research (Switzerland) and The Victorian Institute of Animal Science (Australia), resulted in 49% to 69% protection (Morrison et al. 1996).
Intradermal/subcutaneous immunization with recombinant S. mansoni fatty acid-binding protein Sm14 in FCA, provided complete protection against Fasciola hepatica challenge in mice (Tendler et al. 1996). PCT International Patent Publication WO 94/09142 suggests the use of proteases having cathepsin L type activity, derived of Fasciola hepatica, in the formulation of vaccines for combating helminth parasites; immunization of rabbits with the purified mature enzyme resulted in rabbit antibodies capable of decreasing the activity of the enzyme in vitro.
However, levels of protection obtained with F. hepatica cathepsin L or hemoglobin in cattle were only 53.7% or 43.5%, respectively (Dalton et al. 1996). Cathepsin L belongs to a family of cysteine proteinases, secreted by all stages of the developing parasite. Cathepsin L from F. hepatica is most active at slightly acid or neutral pH (Dalton and Heffernan, 1989). The functions of this proteinase include disruption of host immune function by cleaving host immunoglobulin in a papain-like manner (Smith et al. 1993) and preventing antibody mediated attachment of immune effector cells to the parasite (Carmona et al. 1993). Moreover, cathepsin L is capable of degradation of extracellular matrix and basement membrane components (Berasain et al. 1997), and prepares mucosal surface to be penetrated by a parasite indicating that cathepsin L is involved in tissue invasion. Because of its crucial biological functions, cathepsin L proteases are considered important candidates for the development of an anti-parasite vaccine.
Cathepsin L is synthesized as a preproprotein with a 15-amino-acid (“aa”)-long peptide presequence, a 91-aa-long peptide prosequence or proregion and a 220-aa-long poly)peptide enzymatic part. Of cysteine proteinases the preregion is removed immediately after synthesis and the proprotein comprising the proregion and the part that (constitutes the mature enzyme) is transported to the Golgi. Conversion to the mature enzyme and thus conversion to an enzymatically active state, occurs in the lysosomes and could be due to cathepsin D or to autoactivation. In some cases precursors containing the proregion are secreted (North et al. 1990). Cathepsin L itself has a high affinity for a substrate with Arg at the P1 position and a hydrophobic residue (Phe) at the P2 position (Dowd et al. 1994). It also has autocatalytic activity and cleaves off its prosequence before it obtains its mature enzymatic activity. Cathepsin L2 also cleaves peptides containing Pro at the P2 position, and is therefore capable of cleaving fibrinogen and producing a fibrin clot.
Other potential candidates for an anti-fluke vaccine are hemoglobin, isolated from mature Fasciola hepatica (McGonigle and Dalton 1995) and cathepsin L secreted by adult Fasciola hepatica (Smith et al. 1993; Smith et al. 1994; Spithill 1995). Up to now, next to the irradiated Fasciola gigantica metacercariae (Bitakarami 1973) several antigens have been named as potential protein vaccines:                Fasciola hepatica hemoprotein        Fatty acid-binding protein Sm14 from Schistosoma mansoni         Thiol proteases with Cathepsin L-type activity        Glutathion S-transferase extracted from adult Fasciola hepatica         polypeptide from Fasciola species (Gln-Xaa-Cys-Trp-Xaa)        Serin proteases with dipeptidyl peptidase activity        
However, none of these potential candidates have emerged as an effective vaccine against Fasciola hepatica infection, and a large number of questions, such as: at what site in the host is immunity expressed?; against which stage of the parasite is immunity directed?; at which site in the host this immunity is induced?; which immune mechanisms are involved in protection?; which stage of Fasciola hepatica induces protective immunity?; and, last but not least, which antigens induce protection?, need to be answered before a successful vaccine can be developed. It is clear that answering these questions is greatly hampered by the lack of a suitable animal- or challenge model by which parasitic infections can be studied. And even when animal models are available progress can only be slow because of the fact that the parasitic infection in the host under study takes a considerable time to develop while its outcome depends on various factors that relate to the in time changing host-parasite relationship. For instance, although much focus has been directed to proteins, such as proteases, derived from newly excysted juvenile (NEJ) stages of Fasciola hepatica as candidate protective antigens (see, for instance, Tkalcevic et al. 1995), no clear cut identification of truly protective proteins has been foreseen. To the contrary, early developmental stages of Fasciola hepatica display rapid changes in protein and antigen expression during the early stages of infection, and such changes may even assist the parasite to evade the host immune response (Tkalcevic et al., Parasite Immunology 18:139-147, 1996). It has, for instance, been demonstrated that in parasites, proteases are involved in the invasion of host tissues, the evasion of immune attack mechanisms and help provide nutrients for parasite survival.
Thus, both the abundance of possible different proteins or antigens that need to be studied and the lack of suitable challenge models hamper the possible progress that is needed in the development of parasite vaccines. Crucial for progress in parasite vaccines are new methods to measure protective immunity in order to be able to study a variety of candidate protective antigens and to identify new candidate protective antigens. Thus, new animal models are needed that will increase the number of candidate proteins or substances that can be tested in time.